Method of detecting, identifying and counting enteritis vibrio using gene (rpod) sequences encoding rna polymerase sigma 70 factor

ABSTRACT

To prepare high-performance specific gene-amplification primers for detecting, quantitatively determining or identifying  Vibrio parahaeinolyticus  having low possibility of mis-identification and practically sufficient amplification efficiency and amplification specificity.  
     We have determined the nucleotide sequences of rpoD genes encoding RNA polymerase σ70 factor of type strains of the genus  Vibrio  and of stock strains of  Vibrio parahaemolyticus  (strains containing and those not containing toxin gene); elucidated the phylogenetic relation, and identified nucleotides which are characteristic in  Vibrio parahaemolyticus,  thereby enabling the design of probes containing them and having high specificity, and primers for gene amplification having high specificity and excellent amplification efficiency.

DETAILED DESCRIPTION OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the field of identifying andtesting microoraganisms and pathogenic microbes using genes, andparticularly, testing Vibrio parahaemolyticus. The present inventionrelates to a method for rapidly identifying and testing Vibrioparahaemolyticus that causes food poisoning in the summer season, sothat the field of the present invention includes food processing, publichealth and clinical laboratory testing.

[0003] 2, Background Art

[0004] At the site of food hygiene control, Vibrio parahaemolyticus iscurrently tested based on biochemical phenotypes. According to theinspection guidelines for food hygiene, test samples are inoculated andcultured on TCBS agar media so as to obtain the thus formed greenishblue putatively positive colonies which are unable to degradesaccharose, and then various tests to identify the biochemicalproperties are conducted on the greenish blue colonies fordetermination. However, there are many problems in conducting the teststo identify the biochemical properties at the site of food manufacturingcontrol, because the tests require accomplished techniques and a greatdeal of time and labor. In order to correct the defects of the standardtests to identify the biochemical properties, development of a testmethod using genes has been attempted for the purpose of detecting andidentifying Vibrio parahaemolyticus accurately, rapidly and simply.

DISCLOSURE OF INVENTION

[0005] The following test methods using genes for testing Vibrioparahaemolyticus have been developed so far, but they involve variousproblems.

[0006] For example, PCR primers have been developed for detecting Vibrioparahaemolyticus using as a target gyrB gene which encodes DNA gyrase βsubunit (Japanese Patent Application Laying-Open (Kokai) No. 09-252783,Applied and Environmental Microbiology, Vol.64, No.2, P.681-687).However, the primers are prepared by assuming sections of sequenceswhich are different between Vibrio parahaemolyticus strain ATCC 17802and Vibrio alginolyticus strain ATCC17749 as specific, as a result ofsequence analysis conducted on the gyrB gene of each single strain. Inother words, the primers have not been prepared based on theunderstanding of the phylogenetic relation of the genus Vibrio based onthe gyrB gene sequence, so that the range of specificity is unclear.Also reported is a detection method which uses a PCR method using as atarget toxR gene that has been found as a gene controlling a toxin geneof Vibrio cholera and known to be present also in Vibrioparahaemolyticus (Journal of Clinical Microbiology. 1999 Vol. 37 No. 4,p1173-1177). However, similar to the above detection primers using gyrBgene, PCR primers of this example are prepared by comparing the genesequences of each strain of Vibrio parahaemolyticus and Vibrio cholera,which are relatively distant from each other among phyla, and thenassuming as Vibrio parahaemolyticus-specific sequences, sections of thenucleotide sequences that are different between the two strains. Hence,these primers have also not been prepared based on the understanding ofthe phylogenetic relation based on the toxR gene sequence, so that therange of specificity is unclear. On the other hand, there is anotherdetection method noticing a toxin gene of Vibrio parahaemolyticus(Japanese Patent Application Laying Open (Kokai) No. 4-293486). It islong known that Vibrio parahaemolyticus is divided into a type having atoxin (Thermostable direct haemolysin: TDH) which causes hemolysis byboring holes on the membranes of red blood cells (referred to asKanagawa phenomenon), and a type having no such toxin. In addition toTDH, a recently found toxin is TRH (TDH related haemolysin: TRH) whichis very similar to TDH and causes no Kanagawa phenomenon but causesdiarrhea (1988: Infect Immun. Vol. 56, 961-965). PCR primers have beendeveloped for specifically detecting tdh and trh genes encoding eachtoxin that is thought to cause the pathogenicity of Vibrioparahaemolyticus (Japanese Patent Application Laying Open (Kokai) No.4-293486). However, not all constituents of Vibrio parahaemolyticuspossess such a toxin gene. Most constituents of Vibrio parahaemolyticusderived from environment actually possess no such toxin gene. Since thetotal cell count of all constituents of Vibrio parahaemolyticusincluding strains that possess no toxin gene is considered important atthe site of food hygiene control, the inspection guidelines for foodhygiene require testing Vibrio parahaemolyticus regardless of thepresence or absence of toxin. Accordingly, detection methods for a toxingene, which notice toxin-production ability only, have no compatibilitywith standard methods which are based on biochemical examination, sothat such detection methods are inappropriate as a detection andidentification method at the site of food hygiene control. As describedabove, the practical use of currently available primers for detectingand identifying Vibrio parahaemolyticus is insufficient.

[0007] In addition to these detection methods, there have been reportedmethods for detecting a 0.76 Kb DNA fragment (Appl. Environ. Microbiol.61(4):1311-1317) with unknown functions that is specifically present inVibrio parahaemolyticus or hemolytic factors, such as tlh (Thermolabilehaemolysin: Lett Appl Microbiol 1999, Vol.28, 66-70) and σ-VPH (FEMSMicrobiol Lett 1990; 55(3):339-45). However, none of these methods havebeen proved to be able to reliably detect Vibrio parahaemolyticus, andnone of them can be actually used at the site of evaluation.

[0008] As described above, none of the genetic testing methods considersthat bacterial “species” is a population containing genetic diversity,and a nucleotide sequence of a strain assumed to be a member of acertain bacterial population is used as a common sequence or arepresentative sequence of the population for designing PCR primers.However, gene mutation, which is rapidly accumulated by molecularevolution, particularly neutral mutation, being taken intoconsideration, a strain originally to be detected cannot always bedetected because amplification may be inhibited if slight mutation inprimer region decreased applicability as a primer. Moreover, it isfeared that there may be misidentification, because the lack ofconsideration on differences with closely related species byphylogenetic analysis inhibits design of a primer having sufficientamplification specificity, such that an closely related strain that isnot originally a target is detected.

[0009] Thus, the preparation of high-performance, specificgene-amplification primers for detecting, identifying and quantitativelydetermining Vibrio parahaemolyticus having a proven background forspecificity, low possibility of misidentification, and practicallysufficient amplification efficiency and specificity has been required.

[0010] To establish a method for specifically detecting a gene of acertain phylogenetic group of bacteria, it is required to collect andcompare as many as possible nucleotide sequences of an organism group tobe detected, and of an organism group which is phylogenetically close tothe group. In addition, a gene targeted for specific detection isrequired to have a sufficiently different nucleotide sequence to enablediscrimination from the closest relatives. Thus, a target gene must havea sufficiently rapid rate of evolution.

[0011] Further, a gene which is present independently from phylogeny,such as a toxin gene of Vibrio parahaemolyticus, that is spreadhorizontally at a high frequency, cannot be used. A σ70 factor encodedby rpoD gene that is used as a target in the present invention is afactor controlling gene expression in bacteria at the logarithmic phase,and is a protein essential for survival. Therefore the factor isappropriate for phylogenetic analysis of bacteria, because it is rarelyspread horizontally and has an appropriate rate of evolution (Int. J.Syst. Bacteriol. 1998; 48, 813-819, Int. J. Syst. Bacteriol. 1999; 49,87-95).

BRIEF DESCRIPTION OF DRAWINGS

[0012]FIG. 1. Phylogenetic tree of the genus Vibrio based on rpoD genesequence

[0013]FIG. 2 Phylogenetic analysis of food-derived, Vibrioparahaemolyticus-like colonies based on rpoD gene

[0014] Phylogenetic trees constructed by the neighbor-joining method(NJ) after analysis of an approximately 800 bp partial sequence of rpoDgene determined by direct sequencing method. Strains determined asVibrio parahaemolyticus based on analysis of biochemical properties (theprimary and secondary identification tests were performed according tothe inspection guidelines for food hygiene) were described as V.parahaemolyticus. The phyletic group that Vibrio parahameolyticusbelongs to is denoted as V. p, and other phyletic groups of the genusVibrio are classified into C1 to 5 as shown in the figure. The rpoD genesequence of E. coli strain K12 and that of V. cholerae used herein wereaccession Nos. AE000388 and AE004138 of the GenBank database.

[0015]FIG. 3 Information on specificities among the phyla of thenucleotide sequences of rpoD gene of the genus Vibrio

[0016] The figure shows results after determination of the consensussequences of the phyla V. p and C-1 to 3 (shown in FIG. 1), followed byhomology analysis.

[0017] The upper case is V. p (the phylum that Vibrio parahaemolyticusbelongs to); the lower case is the consensus sequence of the phyla C1 to3. A section indicated by small sequential * denotes that it is the samesequence as the above sequence. A section indicated by large  denotesthat it is a nucleotide specific to Vibrio parahaemolyticus. Symbolsindicated are D=A or G or T; H=A or C or T; V=A or C or G; R=A or G; Y=Cor T, K=G or T, M=A or C, S=G or C, W=A or T, and N=A or G or T or C.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] We have already developed a simple method for determiningnucleotide sequences of rpoD gene which encodes RNA polymerase σ70factor by a PCR direct sequencing method (Japanese Patent ApplicationLaying Open (Kokai) No. 8-256798). (Table 1)

[0019] Therefore, using the method, we have determined the nucleotidesequences of rpoD gene of the type strains of the genus Vibrio(purchased from Institute for Fermentation, Osaka (IFO)) and Vibrioparahaemolyticus stock strains (strains containing and those notcontaining toxin gene) as shown in Table 1 above, thereby clarifying thephylogenetic relation. Specifically, test strains shown in Table 1 werecultured for growth on brain/heart infusion media (NISSUI PHARMACEUTICALCo., Ltd) supplemented with 2% NaCl at 35° C. overnight. Chromosome DNAwas extracted from 1 ml of the culture solution using PUREGENE DNAIsolation Kit (Gentra SYSTEMS). Using the extracted DNAs as templatesand rpoD amplification universal primers (s70S :ACgACTgACCCggTACgCATgTAYATgMgNgARATgggNACNgT and s70R:ATAgAAATAACCAgACgTAAgTTNgCYTCNACCATYTCYTTYTT described in JapanesePatent Application Laying Open (Kokai) No. 8-256798), an rpoD genefragment of approximately 800 bp (positions 334-1125 on rpoD genesequence of Escherichia coli strain K-12, corresponding to a region ofpositions 112 to 376 of an amino acid sequence) was amplified by a PCRmethod. Amplification reaction was performed using thermostable DNApolymerase (AmpliTaq Gold: Applied Biosystems) and a GENE MATE thermalcycler (ISC BioExpress). 50 μl of a reaction solution was prepared tocontain 1 μg of DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂,0.01% gelatin, 0.2 mM of each dNTP, 2.5 U AmpliTaq Gold and 1 μM of eachprimer. The reaction condition consisting of activation (95° C. for 10min) with AmpliTaq Gold, 40 cycles of reaction (94° C. for 1 min, 56° C.for 1 min and 72° C. for 1 min), and then elongation reaction (72° C.for 10 min) was performed. The resulting PCR products were subjected to1% agarose gel (Sea Plaque GTG agarose: BioWhittaker MolecularApplications) electrophoresis (0.5×TAE, 100V for 30 min), and thenstained with ethidium bromide for 10 min. The presence of the productwas confirmed under ultraviolet radiation, excised from the gel, andthen purified using Wizard PCR Preps DNA Purification System (Promega),thereby preparing a template for sequence reaction. Sequence reactionwas performed using sequences for sequence reaction (s70sS :ACgACTgACCCggTACgCATgTA and s70sR: ATAgAAATAACCAgACgTAAgTT. described inJapanese Patent Application Laying Open (Kokai) No. 8-256798) previouslyadded to the above universal primers for rpoD gene as primers, ABI PRISMBigDye Terminator Cycle Sequencing Ready Reaction Kit (AppliedBiosystems) and GENE MATE thermal cycler (ISC BioExpress). The reactionsolution was prepared to have a final volume of 20 μl by mixing 20 ng ofDNA, 3.2 pmol of primers and 8 μl of BigDye Terminator Ready ReactionMix. The cycle sequence reaction consisting of heating at 92° C. for 10min, and 25 cycles of 96° C. for 10 sec, 58° C. or 46° C. (correspondingrespectively to s70sS: ACgACTgACCCggTACgCATgTA and s70sR:ATAgAAATAACCAgACgTAAgTT) for 5 sec, and 60° C. for 4 min was performed.For nucleotide sequence analysis, ABI PRISM 310 GENETIC ANALYZER(Applied Biosystems) was used. Multiple alignment analysis was performedfor the obtained nucleotide sequences using Clustal W computer program,and then phylogenetic trees were constructed by the neighbor-joiningmethod (Mol. Biol. Evol. Vol.4, No. 4, 406-425) based on the geneticdistance calculated with PHYLIP computer program package and Kimura's2—parameter model (J. Mol. Evol, (1980) Vol.16, No.2, p.111-20).

[0020] As a result, it was shown that all the strains determined asVibrio parahaemolyticus containing toxin genes (tdh or trh) and a typestrain (IFO 12711^(T)) together compose an independent mono phyleticgroup, in the strains of the genus Vibrio (see FIG. 1). Therefore, itwas suggested that constituents of bacteria belonging to this phylumshould be determined as Vibrio parahaemolyticus. Next, for 64 strainswhich had been isolated from food samples and form Vibrioparahaemolyticus-like greenish blue colonies on TCBS agar media (NISSUIPHARMACEUTICAL CO., LTD), the nucleotide sequences of rpoD gene weredetermined by procedures similar to those used for the type strains, andthen phylogenetic analysis was conducted. FIG. 2 shows the results. 11out of 64 strains analyzed belonged to the phylum that should bedetermined as the above Vibrio parahaemolyticus. To verify the resultsof the gene analysis, biochemical properties were identified byperforming examination according to the primary and secondaryidentification tests described in the guidelines for food inspection.Specifically, the primary identification test was performed for each ofthe parameters of oxidase, lactose/saccharose degradation ability usingTSI media, gas generation ability upon glucose degradation, hydrogensulfide production ability, indole production ability using SIM media,indole pyruvic acid production ability, confirmation of motility, lysinedecarboxylase activity, Voges-Proskauer test and salt-tolerance (0, 3, 7and 10% NaCl); the secondary identification test was performed for eachof the parameters of ONPG, ornithine decarboxylase activity, argininedehydrase activity and fermentation ability for various sugars(arabinose, maltose, inosit, xylose, salicin, mannite, mannose, lactoseand saccharose). In the primary identification test, Vibrioparahaemolyticus was shown to have the following properties; oxidase,positive; lactose/saccharose degradation in TSI media, no degradation;hydrogen sulfide, no production; gas generation upon glucosedegradation, none; indole production ability on SIM media, positive;indolepyruvic acid production ability, negative; motility, positive;lysine decarboxylase activity, positive; Voges-Proskauer test, negative;and salt-tolerance (0, 3, 7 and 10% NaCl): −, +, + and −. In thesecondary confirmation test, Vibrio parahaeinolyticus was shown to havethe following properties: ONPG, negative; ornithine decarboxylaseactivity, positive; arginine dehydrase activity, negative; sugarfermentation ability (arabinose, positive; maltose, positive; inosit,negative; xylose, negative; salicin, negative; mannite, positive;mannose, positive; lactose, negative and saccharose, negative). Theabove biochemical examination revealed that only 11 strains which hadbeen derived from food and belonged to the phylum that should bedetermined as Vibrio parahaemolyticus according to the previous analysisof rpoD gene were determined as Vibrio parahaemolyticus. Therefore, itwas shown that Vibrio parahaemolyticus groups can be distinguished andidentified accurately by analysis of rpoD gene.

[0021] To establish a genetic screening method capable of detecting onlyVibrio parahaemolyticus constituents, the following procedures wereperformed. First, to clarify differences in nucleotide sequences amongthe closely related species, the nucleotide sequences of rpoD gene ofthe phylum that Vibrio parahaemolyticus belongs to and the neighborphyla were compared, and then positions of nucleotides which areconserved among Vibrio parahaemolyticus constituents but differ fromthose of other bacteria belonging to the genus Vibrio were identified.Specifically, a consensus sequence of phyletic group that Vibrioparahaemolyticus belongs to was determined, while the consensus sequencewas compared with those of the cluster C-1 to C-3, which are otherpyletic groups of bacteria belonging to the genus Vibrio as shown byFIG. 2 to be phylogenetically close to the phylum that Vibrioparahaemolyticus belongs to. Thus, phylogenetically specific informationwas constructed (FIG. 3). It was shown that nucleotide positions 33, 93,102, 123, 141, 147, 148, 192, 198, 204, 223, 229, 234, 243, 259, 261,264, 267, 270, 384, 390, 501, 594, 597, 633, 712, 735 or 798 of SEQ IDNO: 1 in the Sequence Listing are characteristic in the phylum Vibrioparahaemolyticus belongs to. The use of a specific sequence of the thisphylum containing these characteristic nucleotides enables the design ofprobes having high specificity and primers for gene amplification whichhave high specificity and excellent amplification efficiency.

[0022] For example, primers can be designed to always contain aposition(s) at which a nucleotide(s) is different from that of closelyrelated species, using the nucleotide sequence of rpoD gene comprising15 or more consecutive nucleotides containing a nucleotide(s) differentfrom that of the closely related species, preferably, 20 nucleotides ormore, and further preferably, 20 nucleotides or more and 40 nucleotidesor less. Similarly, probes can be designed to always contain aposition(s) at which a nucleotide(s) is different from that of closelyrelated species, using the nucleotide sequence of rpoD gene comprising15 or more consecutive nucleotides containing a nucleotide(s) differentfrom that of the closely related species, preferably, 20 nucleotides ormore, and further preferably, 20 nucleotides or more and 100 nucleotidesor less.

[0023] Further, regions that can be preferably used to prepare theprimers and probes contain at a high frequency the above nucleotide(s)different from that of closely related species, for example, a regioncontaining 259, 261, 264, 267 and 270; a region containing 141, 147 and148; a region containing 192, 198 and 204: a region containing 223, 229and 234; and a region containing 594 and 597. For primers, the 3′terminus is preferably a nucleotide specific to Vibrio parahaemolyticus.

[0024] The gene amplification primers of present inventions may be usedfor detecting, quantitatively determining or identifying Vibrioparahaemolyticus. The present invention encompasses a kit for detecting,quantitatively determining or identifying Vibrio parahaemolyticus whichcomprises these primers and probes and other reagents in combination.TABLE 1 List of Strains Used Type strains of genus Vibrio  8 strains V.parahaemolyticus IFO 12711 T V. alginolyticus IFO 15630 T V.proteolyticus IFO 13287 T V. nereis IFO 15637 T V. campbellii IFO 15631T V. harveyi IFO 15634 T V. carchariae IFO 15632 T V. tubiashii IFO15644 T Stock Strains of Derived from Human V89-655 O3:K6 TRH:+ Vibrioparahaemolyticus 13 strains V89-056 O4:KB TDH:+ V99-157 O1:K56 TDH:+V99-161 O4:K11 TDH:+ V99-177 O4:K8 TDH:+ V99-215 O3:K6 TDH:+ V99-223O4:K9 TDH:+ Derived from Food No toxin production: 6 strains Isolatedstrains on TCBS media 64 strains Derived from food Total 85 strains

EXAMPLES

[0025] The present invention will now be further described by means ofexamples. However, the examples represent embodiments of the presentinvention, and are not intended to limit the invention.

[0026] Specific detection of Vibrio parahaemolyticus was attempted by aPCR method using primers for detecting and identifying Vibrioparahaemolyticus shown in Table 2. In addition, primers described inclaims 10 to 15 are F1, F2, F5, F6, R1 and R2, respectively. ChromosomeDNAs extracted from the test strains shown in Table 1 above were used astemplates. Table 2 shows details about primers used. Amplificationreaction was performed using thermostable DNA polymerase (AmpliTaq Gold:Applied Biosystems) and a GENE MATE thermal cycler (ISC BioExpress). Areaction solution was prepared to have a final volume of 20 μl andcontain DNA 0.1 μg, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂,0.01% gelatin, dNTP (0.2 mM each), 2.5 U of AmpliTaq Gold and primers(see Table 3 for concentration). A reaction condition consisted ofactivation with AmpliTaq Gold (95° C. for 10 min); 35 to 40 reactioncycles of 94° C. for 1 min, annealing for 1 min (see Table 3 fortemperature), and 72° C. for 1 min; and then elongation reaction at 72°C. for 10 min was performed. Table 3 shows primer combinations andamplification reaction conditions. 5 μl of the reaction solution afteramplification was subjected to 2% agarose gel (agarose S: NIPPON GENECO., LTD.) electrophoresis (0.5×TAE, 100V for 30 min), and then stainedwith ethidium bromide for 10 min, followed by confirmation of thepresence or absence of amplified rpoD genes under ultraviolet radiation.

[0027] Using 4 types of sense primers and 2 types of antisense primers,that is, 8 combinations of primers in total, DNAs of the test strainswere screened. Thus, only the DNAs belonging to the phylum that shouldbe determined as Vibrio parahaemolyticus were positive (Table 4). TABLE2 Primers for specific detection of Vibrio parahaemolyticus PrimerSequence Length Position^(a) Direction F1 agarcttcgtctgactgatt 20129-148 Sense F2 aagaagacctagaagatgat 20 251-270 Sense F5cagcwgcgccaaccgcgact 20 185-204 Sense F6 ctgarctgtctgaarctcaa 20 215-234Sense R1 gttaccagtgaatagggca 19 609-591 Antisense R2attcgttaccagtgaatagg 20 613-594 Antisense

[0028] TABLE 3 PCR conditions for primers for specific detection ofVibrio parahaemolyticus PCR conditions Amplified Annealing Number PrimerSense Antisense product temperature of concentration primer primer (bp)(° C.) cycles (mM) F1 R1 483 58 35 0.1 F2 R1 361 56 40 0.1 F5 R1 427 6440 0.1 F6 R1 397 60 35 0.1 F1 R2 485 58 40 0.1 F2 R2 365 60 40 0.5 F5 R2431 60 40 0.1 F6 R2 401 56 40 0.1

[0029] TABLE 4 PCR result using primers for specific detection of Vibrioparahaemolyticus PCR Sequence Strain Kanagawa F1 & F2 & F5 & F6 & numberCluster Name name phenomenon R1 R1 R1 R1 F1 & R2 F2 & R2 F5 & R2 F6 & R21 V. p V. parahaemolyticus V99-161 + + + + + + + + + 2 V. p V.parahaemolyticus IFO 12711 T − + + + + + + + + 3 V. p V.parahaemolyticus V5 − + + + + + + + + 4 V. p V. parahaemolyticus V99-10− + + + + + + + + 5 V. p V. parahaemolyticus V73 − + + + + + + + + 6 V.p V. parahaemolyticus V99-55 − + + + + + + + + 7 V. p V.parahaemolyticus V99-78 − + + + + + + + + 8 V. p V. parahaemolyticus V20− + + + + + + + + 9 V. p V. parahaemolyticus V17 − + + + + + + + + 10 V.p V. parahaemolyticus V47 − + + + + + + + + 11 V. p V. parahaemolyticusV99-223 + + + + + + + + + 12 V. p V. parahaemolyticus V89-655− + + + + + + + + 13 V. p V. parahaemolyticus V48 − + + + + + + + + 14V. p V. parahaemolyticus V99-215 + + + + + + + + + 15 V. p V.parahaemolyticus V46 − + + + + + + + + 16 V. p V. parahaemolyticus V72− + + + + + + + + 17 V. p V. parahaemolyticus V83 − + + + + + + + + 18V. p V. parahaemolyticus V81 − + + + + + + + + 19 V. p V.parahaemolyticus V78 − + + + + + + + + 20 V. p V. parahaemolyticus V84− + + + + + + + + 21 V. p V. parahaemolyticus V69 − + + + + + + + + 22V. p V. parahaemolyticus V99-157 + + + + + + + + + 23 V. p V.parahaemolyticus V89-056 + + + + + + + + + 24 V. p V. parahaemolyticusV99-15 + + + + + + + + 25 V. p V. parahaemolyticusV99-177 + + + + + + + + + 26 C-2 V. harveyi IFO 15634 T − − − − − − − −− 27 C-2 Vibrio spp. V33 − − − − − − − − − 28 C-2 V. carchariae IFO15632 T − − − − − − − − − 29 C-2 Vibrio spp. V80 − − − − − − − − − 30C-2 Vibrio spp. V74 − − − − − − − − − 31 C-2 Vibrio spp. V15 − − − − − −− − − 32 C-2 Vibrio spp. V3 − − − − − − − − − 33 C-2 Vibrio spp. V42 − −− − − − − − − 34 C-2 Vibrio spp. V44 − − − − − − − − − 35 C-2 Vibriospp. V41 − − − − − − − − − 36 C-2 Vibrio spp. V89 − − − − − − − − − 37C-2 Vibrio spp. V87 − − − − − − − − − 38 C-2 Vibrio spp. V76 − − − − − −− − − 39 C-2 Vibrio spp. V88 − − − − − − − − − 40 C-2 Vibrio spp. V4 − −− − − − − − − 41 C-2 Vibrio spp. V86 − − − − − − − − − 42 C-2 V.campbelli IFO 15631 T − − − − − − − − − 43 C-2 Vibrio spp. V18 − − − − −− − − − 44 C-1 Vibrio spp. V68 − − − − − − − − − 45 C-1 Vibrio spp. V85− − − − − − − − − 46 C-1 Vibrio spp. V16 − − − − − − − − − 47 C-1 V.alginolyticus IFO 15630 T − − − − − − − − − 48 C-3 Vibrio spp. V70 − − −− − − − − − 49 C-3 Vibrio spp. V71 − − − − − − − − − 50 C-5 Vibrio spp.V77 − − − − − − − − − 51 C-5 Vibrio spp. V82 − − − − − − − − − 52 C-4Vibrio spp. V45 − − − − − − − − − 53 C-4 Vibrio spp. V8 − − − − − − − −− 54 C-4 Vibrio spp. V10 − − − − − − − − − 55 C-4 Vibrio spp. V13 − − −− − − − − − 56 C-4 Vibrio spp. V6 − − − − − − − − − 57 C-4 Vibrio spp.V9 − − − − − − − − − 58 C-4 Vibrio spp. V7 − − − − − − − − − 59 C-4Vibrio spp. V14 − − − − − − − − − 60 C-4 Vibrio spp. V11 − − − − − − − −− 61 C-4 Vibrio spp. V12 − − − − − − − − − 62 C-4 Vibrio spp. V79 − − −− − − − − − 63 C-4 Vibrio spp. V43 − − − − − − − − − 64 C-4 V. tubiashiiIFO 15644 T − − − − − − − − − 65 C-4 V. nereis IFO 15637 T − − − − − − −− − 66 C-4 Vibrio spp. V64 − − − − − − − − − 67 C-4 Vibrio spp. V56 − −− − − − − − − 68 C-4 Vibrio spp. V55 − − − − − − − − − 69 C-4 Vibriospp. V57 − − − − − − − − − 70 C-4 Vibrio spp. V63 − − − − − − − − − 71C-4 Vibrio spp. V61 − − − − − − − − − 72 C-4 Vibrio spp. V65 − − − − − −− − − 73 C-4 Vibrio spp. V60 − − − − − − − − − 74 C-4 Vibrio spp. V67 −− − − − − − − − 75 C-4 Vibrio spp. V66 − − − − − − − − − 76 C-4 Vibriospp. V59 − − − − − − − − − 77 C-4 Vibrio spp. V58 − − − − − − − − − 78C-4 Vibrio spp. V62 − − − − − − − − − 79 C-4 Vibrio spp. V19 − − − − − −− − − 80 C-4 V. proteolyticus IFO 13287 T − − − − − − − − − 81Shewanella spp. V51 − − − − − − − − − 82 Shewanella spp. V49 − − − − − −− − − 83 Shewanella spp. V53 − − − − − − − − − 84 Shewanella spp. V50 −− − − − − − − − 85 Shewanella spp. V52 − − − − − − − − −

[0030] Industrial Applicability

[0031] The rpoD gene primers and probes of the present invention areexcellent in terms of detection accuracy because they have been designedbased on a thorough understanding of the phylogenetic relation withVibrio parahaemolyticus, by which improvement of the specificity hasbeen studied. Therefore, the rpoD gene primers and probes are of greatadvantage for direct detection under conditions where bacteria are notisolated from food, and allied bacterial species exist together.

[0032] Free Text in Sequence Listing

[0033] SEQ ID No.s 9-12 are primers.

[0034] Present specification incorporates by reference contents ofspecification including claims and drawings of patent application number2001-235806 filed in the Japan Patent Office on Aug. 3, 2001 on whichpriority is claimed.

1 12 1 807 DNA Vibrio parahaemolyticus 1 actcgcraag gcgaaatcgacatcgcaaaa cgcattgaag aaggtattaa ccaagttcaa 60 tcgtctgttg ctgaataccctggcactatt ccttacatcc tagagcaatt tgataargtt 120 caggcagaag arcttcgtctgactgattta atctctggct ttgtagatcc tgacgctgac 180 gatacagcwg cgccaaccgcgactcacatc ggttctgarc tgtctgaarc tcaattagaa 240 gaggaagacg aagaagacctagaagatgat gaagaragcg atgacgattc agatgaytcr 300 gaagaagatg taggtattgayccagagctr gcgcttgaga aattcaacca gctacgcagc 360 acataccaaa atcttcagctagcgatcaat gagtacggct acgacagccc gaaagcaacc 420 gttgctaacg aaatgatgctrgacgtattc aaagaattcc gtctaacacc aaaacagttc 480 gaccacctag tgaacgaacttcgyacwgca atggatcgcg ttcgtactca agaacgtttg 540 atcatgaagt ctgtggttgaatacggcaaa atgccgaaga aatcgttyat tgccctattc 600 actggtaacg aatcaagtgatgcatggcta gacgagatcc tmgcatctga caagccatac 660 gctgagaaaa tcaaacgtaacgaagaagag atccgtcgtt caatckckaa gttaaaaatg 720 attgaagaag agacatmtctaaacgtacar aacattaaag acatcagccg tcgcatgtct 780 atcggtgaag cgaaagcacgccgtgcg 807 2 807 DNA Artificial Sequence Description of ArtificialSequence Consensus sequence among Vibrio spp. other than Vibriohaemolyticus 2 actcgcgaag gcgaaatcga catcgcaaaa cgtattgaag aaggtattaaccaagttcaa 60 tcgtctgttg ctgaataccc wggyacdaty ccrtacatyc ttgagcartttgayaargtw 120 caagcwgaag aaytdcgtct wacwgacctw atytcwgght ttgthgayccwgaygchgay 180 gayacvrcwg chccracrgc racrcacaty ggttctgarc tractgaatctcagytagaa 240 gawgaagayg awgaagacgt tgatgamgac gaagawrgyg aygayrrykmwgatgahdcw 300 gargaagatg twggtatyga yccwgarytd gcgctwgaga aattcaaccarctacgcagy 360 acvtaycaaa aycttcaryt wgcaatcaac garyacggyt ayravagycckaaagcracm 420 gtwgcwaayg arwtgatgct agacgtatty mrmgarttyc gtctracrccwaaacagtty 480 gaycacytag traaygaayt rcgyacnkcd atggatcgyg ttcgtacwcaagarcgyytg 540 atcatgaark cwryngttga atacggcaaa atgccgaaga aatcrttyatygcrctgtty 600 acwggyaayg aatcwashga wgcwtggytr gatgarrtyy twkcwtcwgayaagccatac 660 gctgaraara tyaaacgtar cgaagaagar atycgycgyt caatcrctaarctaaaratg 720 attgarrahg aracytmtct rammgtwcar aacatyaaag acatcagccgtcgyatgtmt 780 atcggtgaag craaagmkcg ycgtgck 807 3 20 DNA Vibrioparahaemolyticus 3 agarcttcgt ctgactgatt 20 4 20 DNA Vibrioparahaemolyticus 4 aagaagacct agaagatgat 20 5 20 DNA Vibrioparahaemolyticus 5 cagcwgcgcc aaccgcgact 20 6 20 DNA Vibrioparahaemolyticus 6 ctgarctgtc tgaarctcaa 20 7 19 DNA Vibrioparahaemolyticus 7 gttaccagtg aatagggca 19 8 20 DNA Vibrioparahaemolyticus 8 attcgttacc agtgaatagg 20 9 44 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 acgactgacc cggtacgcatgtayatgmgn garatgggna cngt 44 10 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 10 acgactgacc cggtacgcat gta 23 11 44 DNAArtificial Sequence Description of Artificial Sequence Primer 11atagaaataa ccagacgtaa gttngcytcn accatytcyt tytt 44 12 23 DNA ArtificialSequence Description of Artificial Sequence Primer 12 atagaaataaccagacgtaa gtt 23

1. A fragment of a gene represented by SEQ ID NO: 1 that can be used fordesigning specific gene-amplification primers or probes, which comprisesany nucleotides, unique to the groups of Vibrio parahaemolyticus, ofposition Nos. 33, 93, 102, 123, 141, 147, 148, 192, 198, 204, 223, 229,234, 243, 259, 261, 264, 267, 270, 384, 390, 501, 594, 597, 633, 712,735 and 798 in the gene (rpoD) encoding RNA polymerase σ70 factor of SEQID NO: 1 in the Sequence Listing.
 2. A gene amplification primer, whichcomprises a strand consisting of 15 or more consecutive nucleotidescontaining any nucleotides, unique to the groups of Vibrioparahaemolyticus, of position Nos. 33, 93, 102, 123, 141, 147, 148, 192,198, 204, 223, 229, 234, 243, 259, 261, 264, 267, 270, 384, 390, 501,594, 597, 633, 712, 735 and 798 in the gene (rpoD) encoding RNApolymerase o70 factor of SEQ ID NO: 1 in the Sequence Listing, or acomplementary strand corresponding thereto.
 3. The gene amplificationprimer of claim 2, which uses a region containing at a high frequencypositions unique to Vibrio parahaemolyticus as specified in claim
 2. 4.The gene amplification primer of claim 3, which comprises a strandcontaining nucleotides of position Nos. 259, 261, 264, 267 and 270 ofSEQ ID NO: 1 in the Sequence Listing, or a complementary strand thereto.5. The gene amplification primer of claim 3, which comprises a strandcontaining nucleotides of position Nos. 141, 147 and 148 of SEQ ID NO: 1in the Sequence Listing, or a complementary strand thereto.
 6. The geneamplification primer of claim 3, which comprises a strand containingnucleotides of position Nos. 192, 198 and 204 of SEQ ID NO: 1 in theSequence Listing, or a complementary strand thereto.
 7. The geneamplification primer of claim 3, which comprises a strand containingnucleotides of position Nos. 223, 229 and 234 of SEQ ID NO: 1 in theSequence Listing, or a complementary strand thereto.
 8. The geneamplification primer of claim 3, which comprises a strand containingnucleotides of position Nos. 594 and 597 of SEQ ID NO: 1 in the SequenceListing, or a complementary strand thereto.
 9. The gene amplificationprimer of claim 2, wherein a nucleotide at the 3′ terminus is a Vibrioparahaemolyticus-unique nucleotide having a position number specified inclaim
 2. 10. The gene amplification primer of claim 2, which comprises5′-agarcttcgtctgactgatt-3′ (SEQ ID NO: 3), or a complementary strandcorresponding thereto.
 11. The gene amplification primer of claim 2,which comprises 5′-aagaagacctagaagatgat-3′ (SEQ ID NO: 4), or acomplementary strand corresponding thereto.
 12. The gene amplificationprimer of claim 2, which comprises 5′-cagcwgcgccaaccgcgact-3′ (SEQ IDNO: 5), or a complementary strand corresponding thereto.
 13. The geneamplification primer of claim 2, which comprises5′-ctgarctgtctgaarctcaa-3′ (SEQ ID NO: 6), or a complementary strandcorresponding thereto.
 14. The gene amplification primer of claim 2,which comprises 5′-gttaccagtgaatagggca-3′ (SEQ ID NO: 7), or acomplementary strand corresponding thereto.
 15. The gene amplificationprimer of claim 2, which comprises 5′-attcgttaccagtgaatagg-3′ (SEQ IDNO: 8), or a complementary strand corresponding thereto.
 16. A methodfor detecting, quantitatively determining or identifying Vibrioparahaemolyticus, which uses the primer of any one of claims 2 to 15.17. A kit comprising the primer of any one of claims 2 to
 15. 18. Aprobe for detecting, quantitatively determining or identifying Vibrioparahaemolyticus, which comprises 15 or more consecutive nucleotidescontaining any nucleotides, unique to the groups of Vibrioparahaemolyticus, of position Nos. 33, 93, 102, 123, 141, 147, 148, 192,198, 204, 223, 229, 234, 243, 259, 261, 264, 267, 270, 384, 390, 501,594, 597, 633, 712, 735 and 798 in the gene (rpoD) encoding RNApolymerase σ70 factor of SEQ ID NO: 1 in the Sequence Listing.